Layer by layer coated mesh for local release of bio-active proteins

ABSTRACT

The present invention relates to biomaterials coated with an active agent eluting coating, wherein implantation of the coated biomaterial results in reduced implant-related complications and/or improved integration of the biomaterial into the host tissue and further relates to kits containing the coated biomaterial. The present invention also relates to methods and kits for coating the biomaterial. It is based, at least in part, on the discovery that biomaterial coated with a cytokine eluting coating resulted in the shift of early stage macrophage polarization that were associated with positive long-term effects such as minimized capsule formation and improved tissue quality and composition as compared to uncoated biomaterials.

2. CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.16/741,269, filed on Jan. 13, 2020, which is a continuation of U.S.patent application Ser. No. 15/459,993, filed on Mar. 15, 2017, now U.S.Pat. No. 10,576,187, which claims priority to U.S. ProvisionalApplication No. 62/308,574, filed Mar. 15, 2016, the contents of whichare hereby incorporated by reference herein and from which priory isclaimed.

1. GRANT INFORMATION

This invention was made with government support under Grant Nos.HD043441 and GM107882 awarded by the National Institute of Health. Thegovernment has certain rights in the invention.

3. INTRODUCTION

The present invention relates to biomaterials coated with an activeagent eluting coating, wherein implantation of the coated biomaterialresults in modulation of the local immune reaction and reducedimplant-related complications and/or improved integration of thebiomaterial into the host tissue.

4. BACKGROUND OF THE INVENTION

The interaction of the host immune system with implantable materials hashistorically been considered to be a negative occurrence associated withdownstream encapsulation and/or implant failure; however, it is nowincreasingly recognized that host-implant interactions can play bothpositive and negative roles following placement. In particular,macrophages have been described as key mediators of the host-implantinteraction and critical determinants of downstream integration andfunctionality.

The host response to implanted materials begins immediately uponintroduction of the material into the host tissue and encompassesmultiple overlapping phases including injury, protein adsorption, acuteinflammation, chronic inflammation, foreign body reaction, granulationtissue formation and eventual encapsulation (1). It is recognized thatthe early interactions which occur at the material-tissue interfacerepresent the initiating events which drive subsequent paracrine andautocrine processes of the host response and subsequent tissueremodeling with significant implications for downstream performance.Recently, macrophage-implant interactions in particular have receivedconsiderable attention as a primary determinant of the outcome ofbiomaterials placement (2-7). A spectrum of macrophage phenotypescontained between two extremes has been identified, ranging frompro-inflammatory (M1) to anti-inflammatory/regulatory (M2) phenotypes,with significant implications in disease, tissue remodeling followinginjury, and biomaterial performance (3, 8-12). Materials which elicitimproved or regenerative remodeling outcomes are associated with a shiftfrom an initially M1 to a more M2 profile during the early stages of theinflammatory response which follows implantation (13-19). Therefore,there is a need in the art for implantable biomaterial that directlymodulates the early-stage macrophage response against the biomaterial inorder to promote downstream tissue integration and functional remodelingin the long-term.

5. SUMMARY OF THE INVENTION

The present invention relates to biomaterials coated with an activeagent eluting coating, wherein implantation of the coated biomaterialresults in modulation of the local immune reaction and reducedimplant-related complications and/or improved integration of thebiomaterial into the host tissue and further relates to kits containingthe coated biomaterial. The present invention also relates to methodsand kits for coating the biomaterial. It is based, at least in part, onthe discovery that biomaterial coated with a cytokine eluting coatingresulted in the shift of early stage macrophage polarization that wasassociated with positive long-term effects such as minimized capsuleformation and improved tissue quality and composition as compared touncoated biomaterials.

The present invention provides coated biomaterials. In certainembodiments, the biomaterials are coated with at least one polycationlayer, at least one polyanion layer, and at least one active agentcontaining layer. In certain embodiments, the active agent is releasedfrom the coating and polarizes macrophages to an M2 phenotype. Incertain embodiments, the biomaterial can be, but is not limited to,mesh, sutures, wound dressings, intraocular lenses, decellularizedmatrices, bone plates, joint replacements, biosensors, catheters,pacemakers, artificial organs, stents, ventricular assist devices, andneural electrodes.

In certain non-limiting embodiments, the polycation (which can becomprised of one or more species of cation) can be, but is not limitedto, one or more polysaccharide (e.g., chitosan), one or more protein(e.g., collagen), a synthetic polyamine, or positively charged polymersor copolymers. In certain non-limiting embodiments, the polyanion (whichcan be comprised of one or more species of anion) can be, but is notlimited to, glycosaminoglycan (e.g., dermatan, dermatan sulfate,hyaluronate, an alginate, chondroitin sulfate, heparan sulfate, or anycombination thereof or negatively charged polymers or copolymers (e.g.,polyacrylates, polyesters, polyurethanes). In certain non-limitingembodiments, the active agent can be a cytokine (e.g., IL-4, IL-13,IL-10, TGF-β, HGF or combinations thereof, or one or more glucocorticoidor combination of glucocorticoids). In certain non-limiting embodiments,the active agent is released to provide an effective concentration ofthe active agent locally at the tissue-implant interface, e.g., atdistances of from about 10 μm to about 100 μm, or up to about 50 μm fromthe coated biomaterial.

In certain non-limiting embodiments, the coating on the biomaterial canhave a thickness ranging from 0.5 nm to a 500 μm. In certainembodiments, the coated biomaterial comprises a total of about 10 toabout 1000 of alternating polycation and polyanion bilayers. In certainnon-limiting embodiments, the alternating polycation and polyanionlayers do not contain an active agent. In certain non-limitingembodiments, the alternating polycation and polyanion layer coatedbiomaterial is coated with at least one active agent containing layer ontop of the alternating polycation and polyanion layers. In certainnon-limiting embodiments, the coated biomaterial comprises about 10 toabout 1000 active agent containing layers. In certain non-limitingembodiments, the coated biomaterial comprises about 40 active agentcontaining layers on top of about 10 alternating polycation andpolyanion bilayers.

The present invention provides a method for coating a biomaterial. Incertain non-limiting embodiments, the biomaterial is negatively charged.In certain non-limiting embodiments, the biomaterial has a net negativeor a net positive charge. In certain non-limiting embodiments, thenegatively charged biomaterial is coated with a polycation layer. Incertain non-limiting embodiments, the biomaterial is first treated toinduce either a positive or negative surface charge to facilitatecoating. In certain non-limiting embodiments, the polycation layer iscoated with a polyanion layer. In certain non-limiting embodiments, thebiomaterial is coated with alternating polycation and polyanion layers.In certain non-limiting embodiments, once the biomaterial is coated withalternating polycation and polyanion layers, the coated biomaterial iscoated with at least one layer containing at least one active agent. Incertain non-limiting embodiments, the coated biomaterial is sterilized.

6. BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 illustrates a Layer by Layer coating procedure performed onpolypropylene surgical meshes.

FIG. 2A provides x-ray photoelectron spectroscopy spectra (XPS) of LbLcoated (dark gray), RFGD treated (gray) and pristine (light gray) mesh.

FIG. 2B provides images of alcian blue stained 1 cm² pieces of pristine(i), RFGD treated (ii) and LbL coated meshes (iii).

FIGS. 3A-3H provide scanning electron microscopy images of meshes, whereFIGS. 3A-3D provide images at 40× FIGS. 3E-3H provide images at 150×.FIGS. 3A and 3E show pristine meshes; FIGS. 3B and 3F show RFGD treatedmeshes; FIGS. 3C and 3G show LbL coated meshes; and FIGS. 3D and 3H showIL-4 loaded [40B] meshes. In FIGS. 3A-3H, scale bars represent 200 μm.

FIGS. 4A-4B provide attenuated total reflectance—Fourier transforminfrared (ATR-FTIR) spectra of pristine (blue), RFGD treated (red) andLbL coated (green) meshes from wavelengths 2800-3700 cm⁻¹ (FIG. 4A) and1300-1800 cm⁻¹ (FIG. 4B).

FIG. 5A provides confocal microscopy images of IL-4 immunolabeledpolypropylene fibers of pristine (i), coated [no IL-4] (ii) and IL-4loaded [40B] (iii) mesh. Scale bars represent 100 μm.

FIG. 5B provides cumulative release of IL-4 (nanograms) versus time(days) from 1 cm² pieces of IL-4 loaded mesh (20, 40 and 60 bilayers).Coated (no IL-4) mesh was used as a control. Power law dependence curvesalong with equations and coefficients are presented.

FIG. 6 shows the log-log linear fittings of IL-4 cumulative releaseversus time.

FIG. 7A illustrates arginase-1 immunolabelled bone marrow-derivedmacrophage cultures exposed to 1 cm² pieces of pristine, coated (noIL-4) and IL-4 loaded (40B) meshes for 72 hrs. IL-4 (20 ng/mL) was usedas positive control. Scale bars represent 100 μm.

FIG. 7B shows the concentration of IL-4 released by IL-4 loaded meshes(40 bilayers) for 72 hours. Bars represent the mean±SEM.

FIG. 8A provides CellProfiler image analysis from arginase-1immunolabeled macrophages in an in-vitro culture exposed to 1 cm² piecesof pristine, coated [no IL-4] and IL-4 loaded [40B] mesh. Isotype andIL-4 [20 ng/mL] were used as negative and positive controls,respectively.

FIG. 8B shows the number of arginase-1 positive macrophages determinedfrom the CellProfiler analysis of FIG. 8A. Bars represent the mean±SEM.(*) Statistically significant (p<0.05), using one-way ANOVA and Tukey'stest. (ns) Non-significant.

FIG. 9 shows H&E stained tissue sections at 10× from mice implanted witha 1 cm² piece of pristine, coated (no IL-4) and IL-4 loaded (40B) meshat 7 days (top panel) and 14 days (bottom panel). Healthy and SHAM (nomesh surgery) were used as controls. Scale bars represent 200 μm.

FIG. 10A shows arginase-1 immunolabeled (brighter cells) tissue sectionsof mice implanted with a 1 cm² piece of pristine, coated (no IL-4) andIL-4 loaded (40B) mesh for 7 days (top panel) and 14 days (bottompanel). DAPI was used to stain cell nuclei. Scale bars represent 40 μm.

FIG. 10B illustrates the arginase-1/DAPI pixel ratio versus distance ofarginase-1 immunolabeled tissue sections at 7 days.

FIG. 10C provides the arginase-1/DAPI pixel ratio at 50 μm of arginase-1immunolabeled tissue sections at 7 and 14 days. Points and barsrepresent the mean±SEM (N=7-9). (*) Statistically significant (p<0.05),using one-way ANOVA and Tukey's test. (ns) Non-significant.

FIG. 10D shows iNOS immunolabeled (brighter cells) tissue sections ofmice implanted with a 1 cm² piece of pristine, coated (no IL-4) and IL-4loaded (40B) mesh for 7 days (top panel) and 14 days (bottom panel).DAPI was used to stain cell nuclei. Scale bars represent 40 μm.

FIG. 10E illustrates the iNOS/DAPI pixel ratio versus distance of iNOSimmunolabeled tissue sections at 7 days.

FIG. 10F provides the iNOS/DAPI pixel ratio at 50 μm of iNOSimmunolabeled tissue sections at 7 and 14 days. Points and barsrepresent the mean±SEM (N=7-9). (*) Statistically significant (p<0.05),using one-way ANOVA and Tukey's test. (ns) Non-significant.

FIGS. 11A-11D provide image analysis of cells. FIG. 11A shows totalcells (DAPI) and FIG. 11B shows F4/80

cells as percentages of total cells (DAPI) surrounding single meshfibers of tissue cross sections of mice implanted with a 1 cm² piece ofpristine, coated (no IL-4) and IL-4 loaded (40B) mesh for 7 days and 14days. FIG. 11C shows arginine-1 positive F4/80 positive cells and FIG.11D shows iNOS positive F4/80 positive cells as percentages of totalF4/80 positive cells surrounding single mesh fibers of tissue crosssections of mice implanted with a 1 cm² piece of pristine, coated (noIL-4) and IL-4 loaded (40B) mesh for 7 days and 14 days. Bars and pointsrepresent the mean±SEM (N=8). Statistical significance as (*) p<0.05,(**) p<0.01, (***) p<0.001 and (****) p<0.0001, using two-way ANOVA withTukey's (groups) and Sidak's (days) tests. All other differences arenon-significant.

FIG. 12A shows Masson's Trichrome stained tissue sections of miceimplanted with a 1 cm² piece of pristine, coated (no IL-4) and IL-4loaded (40B) mesh at 90 days. Scale bars represent 200 μm.

FIG. 12B provides image analysis of capsule deposition (area %)surrounding mesh fibers (3 images of a single fiber at 20× per sample,N=8 samples). Bars represent the mean±SEM. (*) Statistically significant(p<0.05), using two-way ANOVA and Tukey's test. All other meandifferences are non-significant.

FIG. 12C shows Picro Sirius Red stained tissue sections (20×) of miceimplanted with a 1 cm² piece of pristine, coated (no IL-4) and IL-4loaded (40B) mesh at 90 days. Scale bars represent 100 μm.

FIG. 12D provides image analysis of collagen capsule quality,surrounding mesh fibers (3 images of a single fiber at 20× per sample,N=8). Bars represent the mean±SEM. (*) Statistically significant(p<0.05), using two-way ANOVA and Tukey's test. All other meandifferences are non-significant.

FIG. 13 provides the mean thickness surrounding mesh fibers (3 images ofa single fiber at 20× per sample, N=8 samples). Bars represent themean±SEM. Statistical significance as (**) p<0.01 and (****) p<0.0001,using two-way ANOVA with Tukey's test. All other differences arenon-significant.

7. DETAILED DESCRIPTION OF THE INVENTION

The present invention provides biomaterials coated with an active agenteluting coating, wherein implantation of the coated biomaterial resultsin modulation of the local immune reaction and reduced implant-relatedcomplications and/or improved integration of the biomaterial into thehost tissue and further relates to kits containing the coatedbiomaterial. In certain non-limiting embodiments, the biomaterials arecoated with at least one polycation layer, at least one polyanion layer,and at least one active agent containing layer. In certain non-limitingembodiments, the active agent is released from the coating and polarizesmacrophages to an M2 phenotype. The present invention further providesmethods and kits for coating the biomaterial.

The term “biomaterial,” refers to a material which has properties thatare adequate for mammalian body reconstruction, medical deviceconstruction, and/or drug control/release devices or products. This termincludes absorbable devices and products (e.g., absorbable sutures,absorbable clips, absorbable staples, absorbable pins, absorbable rods(for repairing broken bones), absorbable joints, absorbable vasculargrafts, absorbable fabrics or meshes (e.g., for hernia repair, softtissue patches), absorbable sponges, absorbable adhesives and absorbabledrug control/release devices) as well as non-absorbable devices andproducts, (e.g., implantable repair or support meshes (e.g., for pelvicorgan prolapse, artificial chest wall, artificial thoracic or abdominalwall prosthesis, or hernia support) acetabular or tibia components ofjoint prostheses, and bone cement. The term “absorbable” as used hereinrefers to materials that will be degraded and subsequently absorbed bythe body. The term “non-absorbable” as used herein refers to materialsthat will not be degraded and subsequently absorbed by the body.

The terms “macrophage polarization” or “polarization of macrophages”, asused interchangeably herein, refer to controlling the macrophagemicroenvironment to elicit a particular macrophage phenotype. Polarizedmacrophages can be broadly classified into two main phenotypes: 1) M1,which is pro-inflammatory and 2) M2, which isanti-inflammatory/regulatory. Materials which elicit improved orregenerative remodeling outcomes are associated with a shift from aninitially M1 to a more M2 profile during the early stages of theinflammatory response which follows implantation. Macrophages can bepolarized to M2 by treating the microenvironment with cytokines (e.g.,IL-4, IL-13, IL-10, or combinations thereof) and/or glucocorticoids.

The term “polyelectrolyte layers” refers to coating layers that arecharged. For example, the polyelectrolyte layer can be either apolycation layer or a polyanion layer. A “polyelectrolyte bilayer”refers to combination of a polycation and a polyanion polymer in abilayer.

The term “polycation” refers to any polymer that has a net positivecharge at the pH the layer is formed. Examples of polycations include,but are not limited to, a polysaccharide, a protein, a syntheticpolyamine, or a synthetic polymer or polypeptide. In certainembodiments, polycation polysaccharides bearing one or more amino groupscan be used herein. In certain embodiments, the polycation is thenatural polysaccharide chitosan. As used herein, the term “chitosan”refers to a linear polysaccharide composed of randomly distributed6-(1-4)-linked D-glucosamine (deacetylated unit) andN-acetyl-D-glucosamine (acetylated unit). Chitosan is produced bydeacetylation of chitin. The term “chitosan” relates to chitosan,chitosan derivatives and mixtures of chitosan and chitosan derivatives(e.g., glycol-chitosan, amine-grafted chitosan, fluorescent-taggedchitosan). Similarly, the protein can be synthetic ornaturally-occurring. In certain embodiments, the biodegradable polyamineis a synthetic random copolypeptide, synthetic polyamine such aspoly(β-aminoesters), polyester amines, poly(disulfide amines), mixedpoly(ester and amide amines), and peptide crosslinked polyamines. Thepolycation polymers can be branched, linear, or a combination thereof.

The term “polyanion” refers to any polymer that has a net negativecharge at the pH the layer is formed. Examples of polyanions include,but are not limited to, a polysaccharide, a protein, or a syntheticpolymer or polypeptide. In certain embodiments, the polyanion is apolysaccharide. Examples of polyanion polysaccharides useful hereininclude, but are not limited to, a hyaluronate, an alginate, chondroitinsulfate, dermatan, dermatan sulfate, heparan sulfate, or any combinationthereof. The polyanion polymers can be branched, linear, or acombination thereof.

The terms “tune” or “tunable”, as used herein, means the ability toadjust the number and/or composition of layers of the coated biomaterialto alter the pharmacokinetic distribution of the active agent. Forexample, altering the number and/or composition of layers can alteractive agent release characteristics such as, but not limited to, thedosage, release rates, duration, and distribution of the active agent.Tuning of the number and/or composition of layers of the coatedbiomaterial can be accomplished a number of ways, including but notlimited to varying the number of alternating polycation and polyanionlayers (e.g., increasing or decreasing the number of layers) prior toadding the active agent containing layers; varying the polycation and/orpolyanion used in the alternating polycation and polyanion layers (e.g.change the polycation and/or polyanion used or utilize differentcombinations of polycations and/or polyanions); altering the number ofactive agent containing layers; altering the composition of the activeagent containing layer; adding additional polycation and/or polyanionlayers on top and/or in between the active agent layers.

The term “active agent” refers to an agent that is capable of having aphysiological effect when administered to a subject. In certainembodiments, the term “active agent” refers to an agent that canpolarize macrophages away from the M1 phenotype and/or towards the M2phenotype for example, but not limited to cytokines (e.g., IL-4, IL-13,IL-10, or combinations thereof). and/or glucocorticoids (e.g.,betamethasone, clocortolone, cortisone, dexamethasone, fludrocortisone,fluocortolone, fluprednylidene, hydrocortisone, medrysone,methylprednisolone, paramethasone, prednisolone, prednisone,prednylidene, triamcinolone, triamcinolone acetonide and their esters).

For clarity of description, and not by way of limitation, the detaileddescription of the invention is divided into the following subsections:

(1) coated biomaterial;

(2) methods of coating the biomaterial; and

(3) kits.

7.1. Coated Biomaterials

The present invention provides biomaterials coated with an active agenteluting coating. In certain non-limited embodiments, implantation of thecoated biomaterial results in modulation of the local immune reactionand reduced implant-related complications and/or improved integration ofthe biomaterial into the host tissue as compared to non-coatedbiomaterials or coated biomaterials not containing an active agent. Incertain embodiments, the biomaterials are coated with at least onepolycation layer, at least one polyanion layer, and at least one activeagent containing layer. The number and sequence of layers can bemodified in order to provide the desired amount and release time ofactive agent from the coated biomaterial.

In certain non-limiting embodiments, the active agent is released fromthe coating and polarizes macrophages away from an M1 phenotype and/orto an M2 phenotype.

In certain non-limiting embodiments, implantation of the coatedbiomaterial results in reduced implant-related complications as comparedto non-coated or coated biomaterial not containing and active agent. Forexample, the coated biomaterial results in diminished formation offibrotic capsule surrounding the implant, reduced biomaterial associatedinflammation and tissue degradation, and improved tissue integration.

In certain non-limiting embodiments, implantation of the coatedbiomaterial results in improved integration of the biomaterial into thehost tissue as compared to non-coated biomaterials or coatedbiomaterials not containing an active agent. For example, the coatedbiomaterial results in faster remodeling, faster tissue regenerations,increased functional remodeling of the tissue area, and reducedmorbidities.

7.1.1. Biomaterials

The present invention provides for a coated biomaterial, wherein thebiomaterial can be any material which has properties that are adequatefor mammalian body reconstruction, medical device construction, and/ordrug control/release devices or products as defined above. In certainnon-limiting embodiments, the biomaterial can be, but is not limited to,mesh, sutures, wound dressings, intraocular lenses, decellularizedmatrices, bone plates, joint replacements, biosensors, catheters,pacemakers, artificial organs, stents, and ventricular assist devices.In a non-limiting embodiment, as exemplified below, the biomaterial canbe a mesh for implantable repair or support (e.g., for pelvic organprolapse, artificial chest wall, artificial thoracic or abdominal wallprosthesis, or hernia support).

The biomaterial can be made of any materials that will accept a coating.In non-limiting examples, the biomaterial can be made of polypropylene,polyesthers (e.g., Mercilene, Dacron) ePTFE, which is expandedpolytetrafluoroethylene, polyurethanes, titanium, gold, and/or teflon.

In certain non-limiting embodiments, the biomaterial can be made of amaterial that can hold a charge and/or a net charge. The biomaterial canhold either a positive or negative charge. For example, if thebiomaterial holds a negative charge, the first coating layer next to thebiomaterial should be a positively charged layer, and if the biomaterialholds a positive charge, the first coating layer next to the biomaterialshould be a negatively charged layer.

In certain non-limiting embodiments, the biomaterial can be, but is notlimited to surgical mesh. In certain non-limiting embodiments, the meshcan be used to provide support when repairing weakened or damagedtissue. In certain non-limiting embodiments, the mesh can be used forexample, but not limited to, pelvic organ prolapse. In certainnon-limiting embodiments, the mesh can be a polypropylene mesh. Incertain non-limiting embodiments, the mesh can be a non-absorbentpolypropylene mesh (e.g., Gynemesh PS from Ethicon). In certainnon-limiting embodiments, the mesh can be a canonical mesh. The mesh canbe of any type, shape, size, or material.

The total number of layers of the biomaterial coating should not alterthe architecture or topography of the biomaterial. For example, thebiomaterial coating should not alter the biomaterial shape, size,performance, porosity, or combinations thereof.

In certain embodiments, the coating on the biomaterial can be from about0.5 nm to about 500 μm thick. In certain embodiments, the coating on thebiomaterial can be from about 1 nm to about 400 μm, about 10 nm to about300 μm, about 20 nm to about 200 μm, about 30 nm to about 100 μm, about40 nm to about 50 μm, about 50 nm to about 10 μm, about 60 nm to about 1μm, about 70 nm to about 900 nm, about 80 nm to about 800 nm, about 90nm to about 700 nm, about 100 nm to about 600 nm, about 200 nm to about500 nm, or about 300 nm to about 400 nm thick. In certain embodiments,the coating can be from about 0.3 nm to about 0.8 nm, about 0.4 nm toabout 0.7 nm, or about 0.5 nm to about 0.6 nm in thickness. In certainembodiments, the coating can be from about 1 nm to about 1000 nm, about10 nm to about 900 nm, about 20 nm to about 800 nm, about 30 nm to about700 nm, about 40 nm to about 600 nm, about 50 nm to about 500 nm, about60 nm to about 400 nm about 70 nm to about 300 nm, about 80 nm to about200 nm or about 90 nm to about 100 nm in thickness. In certainembodiments, the coating can be from about 1 μm to about 500 μm, about10 μm to about 400 μm, about 20 μm to about 300 μm, about 30 μm to about200 μm, about 40 μm to about 100 μm, about 50 μm, to about 90 μm, orabout 60 μm to about 80 μm in thickness. In certain embodiments, thecoating is no more than about 0.2 nm, about 0.3 nm, about 0.4 nm, about0.5 nm, about 0.6 nm, about 0.7 nm, about 0.8 nm, about 0.9 nm, or about1 nm in thickness. In certain embodiments, the coating is no more thanabout 2 nm, about 3 nm, about 4 nm, about 5 nm, about 6 nm, about 7 nm,about 8 nm, about 9 nm, about 10 nm, about 20 nm, about 30 nm, about 40nm, about 50 nm, about 60 nm, about 70 nm, about 80 nm, about 90 nm, orabout 100 nm in thickness.

7.1.2. Polyelectrolyte Layers

The present invention provides for a coated biomaterial, wherein thebiomaterial can be coated with polyelectrolyte layers. In certainnon-limiting embodiments, the biomaterial can be coated with alternatingpolycation and polyanion layers (i.e., polyelectrolyte bilayers) (seeFIG. 1 ). The layer closest to the biomaterial can be either apolycation layer or a polyanion layer. The layer closest to the activeagent containing layer can be either a polycation layer or a polyanionlayer. In certain non-limiting embodiments, the polycation and/orpolyanion layers can be distributed among and/or on top of the activeagent containing layers.

In certain non-limiting embodiments, the polyelectrolytes can beantimicrobial. The polycation can be a polysaccharide, a protein, asynthetic polyamine, or a synthetic polymer or polypeptide as discussedabove. The polyanion can be a polysaccharide, a protein, or a syntheticpolymer or polypeptide as discussed above. In certain non-limitingembodiments, as exemplified below, the polycation is chitosan. Chitosanhas known biocompatibility, antimicrobial activity, and is degraded byactivated macrophages. In certain non-limiting embodiments, asexemplified below, the polyanion is dermatan sulfate. Dermatan sulfate(also known as chondroitin sulfate B) plays a role in extracellularmatrix (ECM) regulation and is able to enhance IL-4 bioactivity in-vivo.In certain non-limiting embodiments, the alternating polycation andpolyanion layers can be chitosan-dermatan sulfate alternating layers. Incertain embodiments, the alternating chitosan-dermatan sulfatealternating layers can provide enhanced release and bioactivity of theactive agent (e.g., IL-4) in the context of macrophage mediatedhost-implant interactions.

The total number of alternating polycation and polyanion layers can beadjusted in order to tune the release of the active agent from thecoated biomaterial. In certain embodiments, the bilayer core coatingserves to make the surface charge more solid, consistent, and/or strongfor the deposition of the active agent containing layers.

In certain non-limiting embodiments, the total number of alternatingpolycation and polyanion layers (i.e., polyelectrolyte bilayers) can befrom about 10 to about 1000 bilayers. In certain embodiments, the totalnumber of polyelectrolyte bilayers can be from about 20 to about 900,about 30 to about 800, about 40 to about 700, about 50 to about 600,about 60 to about 500, about 70 to about 400, about 80 to about 300, orabout 90 to about 200. In certain embodiments, the total number ofpolyelectrolyte bilayers can be from about 20 to about 90, about 30 toabout 80, about 40 to about 70, or about 50 to about 60. In certainembodiments, the total number of polyelectrolyte bilayers can be fromabout 6 to about 18 bilayers, about 8 to about 16 bilayers, or about 10to about 14 bilayers. In certain embodiments, the total number ofpolyelectrolyte bilayers can be from about 8 to about 14 bilayers orabout 10 to about 12 bilayers. In certain embodiments, the total numberof polyelectrolyte bilayers can be at least 4 bilayers, at least 6bilayers, at least 8 bilayers, at least 10 bilayers, at least 12bilayers, at least 14 bilayers, at least 16 bilayers, at least 18bilayers, or at least 20 bilayers. In certain embodiments, the totalnumber of polyelectrolyte bilayers can be about 10 bilayers.

The polycation layer can be made of one type of polycation or acombination of different polycations. In certain embodiments, eachpolycation layer contains only one type of polycation. In certainnon-limiting embodiments, the coated biomaterial contains more than onetype of polycation, wherein the different polycations are in the sameand/or different layers.

The polyanion layer can be made of one type of polyanion or acombination of different polyanions. In certain non-limitingembodiments, each polyanion layer contains only one type of polyanion.In certain non-limiting embodiments, the coated biomaterial containsmore than one type of polyanion, wherein the different polyanions are inthe same and/or different layers.

In certain non-limiting embodiments, the polyelectrolyte layer containsadditional excipients known to those of skill in the art.

7.1.3. Active Agent Containing Layer

The present invention provides for a coated biomaterial, wherein thebiomaterial can be further coated with an active agent containing layer.In certain non-limiting embodiments, the biomaterial coated with thealternating polycation and polyanion layers can be further coated withat least one active agent containing layer (see FIG. 1 ). Thepolyelectrolyte layer closest to the active agent containing layer canbe either a polycation layer or a polyanion layer. In certainnon-limiting embodiments, the polyelectrolyte layers can be distributedamong and/or on top of the active agent containing layers.

In certain non-limiting embodiments, the active agent containing layercan include either a polycation or a polyanion. In certain embodiments,if the active agent containing layer holds a negative charge, thepolyelectrolyte layer next to the active agent containing layer shouldbe a polycation layer, and if the active agent containing layer holds apositive charge, the polyelectrolyte layer next to the active agentcontaining layer should be a polyanion layer.

Materials which elicit improved or regenerative remodeling outcomes areassociated with a shift from an initially M1 to a more M2 profile duringthe early stages of the inflammatory response which followsimplantation. In certain embodiments, the active agent is one that canmitigate the foreign body reaction and/or lead to improved implantintegration. For example, the active agent can polarize macrophages awayfrom the M1 phenotype and/or towards the M2 phenotype. In certainembodiments, polarization of the macrophages to the M2 phenotype willmitigate the foreign body reaction and/or lead to improved implantintegration. In a non-limiting embodiment, as exemplified below, theactive agent can polarize macrophages towards the M2 phenotype and awayfrom the M1 phenotype.

The macrophage phenotype can be tested by immunocytochemistry. Forexample, immuno-labeling can be performed to assess the phenotypicprofiles of the cells post-implantation (e.g., 7 and/or 14 days), suchas testing for the presence of arginase-1 (an M2 marker) and/orinducible nitric oxide synthase (iNOS, an M1 marker). Image analysis canbe performed using a custom-designed algorithm (Wolfram Mathematica,Version 10.0) in order to quantify labeling (normalized and expressed ascumulative arginase-1/DAPI pixel ratio) as a function of distance fromthe biomaterial surface.

The total number of active agent containing layers can be adjusted inorder to tune the pharmacokinetic profile of the active agent from thecoated biomaterial. In certain embodiments, the total number of activeagent containing layers can be from about 10 to about 1000 bilayers.Depending on what polyelectrolyte is contained in the active agentlayer, each active agent layer is separated by a polyelectrolyte layerof the opposite charge with or without an active agent. In certainembodiments, the total number of active agent containing layers can befrom about 20 to about 900 layers, about 30 to about 800 layers, about40 to about 700 layers, about 50 to about 600 layers, about 60 to about500 layers, about 70 to about 400 layers, about 80 to about 300 layers,or about 90 to about 200 layers. In certain embodiments, the totalnumber of active agent containing layers can be from about 20 to about90 layers, about 30 to about 80 layers, about 40 to about 70 layers, orabout 50 to about 60 layers. In certain embodiments, the total number ofactive agent containing layers can be from about 10 to about 75 layers,about 15 to about 60 layers, about 20 to about 55 layers, about 25 toabout 50 layers, about 30 to about 45 layers or about 35 to about 40layers. In certain embodiments, the total number of active agentcontaining layers can be from about 20 to about 30 layers, about 20 toabout 40 layers, about 20 to about 50 layers, about 20 to about 60layers, about 30 to about 40 layers, about 30 to about 50 layers, about30 to about 60 layers, about 40 to about 50 layers, about 40 to about 60layers, or about 50 to about 60 layers. In certain embodiments, thetotal number of active agent containing layers can be from about 22 toabout 38 layers, about 24 to about 36 layers, about 26 to about 34layers, about 28 to about 32 layers, about 32 to about 48 layers, about34 to about 46 layers, about 36 to about 44 layers, about 38 to about 42layers, about 42 to about 58 layers, about 44 to about 56 layers, about46 to about 54 layers, or about 48 to about 52 layers. In certainembodiments, the total number of active agent containing layers can beat least 20 layers, at least 21 layers, at least 22 layers, at least 23layers, at least 24 layers, at least 25 layers, at least 26 layers, atleast 27 layers, at least 28 layers, at least 29 layers, at least 30layers, at least 31 layers, at least 32 layers, at least 33 layers, atleast 34 layers, at least 35 layers, at least 36 layers, at least 37layers, at least 38 layers, at least 39 layers, at least 40 layers, atleast 41 layers, at least 42 layers, at least 43 layers, at least 44layers, at least 45 layers, at least 46 layers, at least 47 layers, atleast 48 layers, at least 49 layers, at least 50 layers, at least 51layers, at least 52 layers, at least 53 layers, at least 54 layers, atleast 55 layers, at least 56 layers, at least 57 layers, at least 58layers, at least 59 layers, or at least about 60. In certainembodiments, the total number of active agent containing layers can beabout 20 layers, about 21 layers, about 22 layers, about 23 layers,about 24 layers, about 25 layers, about 26 layers, about 27 layers,about 28 layers, about 29 layers, about 30 layers, about 31 layers,about 32 layers, about 33 layers, about 34 layers, about 35 layers,about 36 layers, about 37 layers, about 38 layers, about 39 layers,about 40 layers, about 41 layers, about 42 layers, about 43 layers,about 44 layers, about 45 layers, about 46 layers, about 47 layers,about 48 layers, about 49 layers, about 50 layers, about 51 layers,about 52 layers, about 53 layers, about 54 layers, about 55 layers,about 56 layers, about 57 layers, about 58 layers, about 59 layers,about 60, about 61, about 62, about 63, about 64, about 65, about 66,about 67, about 68, about 69, about 70, about 71, about 72, about 73,about 74, about 75, about 76, about 7, about 78, about 79, or about 80.

The total number of polyelectrolyte and/or active agent containinglayers can be adjusted in order to tune the release of the active agentfrom the coated biomaterial. For example, the composition of the layerscan be altered such that the dosage, release rates, duration, anddistribution of the active agent can be controlled.

In certain non-limiting embodiments, the polyelectrolyte and/or activeagent containing layers can be adjusted so that the active agent isreleased to provide an effective concentration of the active agent at adistance of about 10 μm to about 100 μm from the coated biomaterial. Incertain embodiments, the layers can be adjusted so that the active agentis released at a distance of about 15 μm to about 95 μm, about 20 μm toabout 90 μm, about 25 μm to about 85 μm, about 30 μm to about 80 μm,about 35 μm to about 75 μm, about 40 μm to about 70 μm, about 45 μm toabout 65 μm, or about 50 μm to about 60 μm from the coated biomaterial.In certain embodiments, the layers can be adjusted so that the activeagent is released at a distance of about 5 μm to about 50 μm, about 10μm to about 45 μm, about 15 μm to about 40 μm, about 20 μm to about 35μm, or about 25 μm to about 30 μm. In certain embodiments, the layerscan be adjusted so that the active agent is released at a distance ofabout 50 μm from the coated biomaterial.

In certain embodiments, the layers can be adjusted so that the activeagent is released to provide an effective concentration of the activeagent at a distance of up to about 100 μm, up about 95 μm, up to about90 μm, up to about 85 μm, up to about 80 μm, up to about 75 μm, up toabout 70 μm, up to about 65 μm, up to about 60 μm, up to about 55 μm, orup to about 50 μm from the coated biomaterial. As used herein, thephrase “effective concentration” means a concentration of the activeagent that is able to polarize macrophages towards the M2 phenotype andaway from the M1 phenotype.

In certain non-limiting embodiments, the polyelectrolyte and/or activeagent containing layers can be adjusted so that the active agent isreleased for about 2 days to about 14 days, about 2 days to about 7days, about 2 days to about 10 days, about 7 days to about 14 days, orabout 7 days to about 10 days. In certain embodiments, thepolyelectrolyte and/or active agent containing layers can be adjusted sothat the active agent is released for at least about 2 days, at leastabout 3 days, at least about 4 days, at least about 5 days, at leastabout 6 days, at least about 7 days, at least about 8 days, at leastabout 9 days, at least about 10 days, at least about 11 days, at leastabout 12 days, at least about 13 days, at least about 14 days, at leastabout 15 days, at least about 16 days, at least about 17 days, at leastabout 18 days, at least about 19 days, or at least about 20 days. Incertain embodiments, the polyelectrolyte and/or active agent containinglayers can be adjusted so that the active agent is released for no morethan about 2 days, no more than about 3 days, no more than about 4 days,no more than about 5 days, no more than about 6 days, no more than about7 days, no more than about 8 days, no more than about 9 days, no morethan about 10 days, no more than about 11 days, no more than about 12days, no more than about 13 days, no more than about 14 days, no morethan about 15 days, no more than about 16 days, at least no more thanabout 17 days, at least no more than about 18 days, at least no morethan about 19 days, or at least no more than about 20 days.

In certain non-limiting embodiments, the biomaterial coating providesfor a delay in the release of the active agent from the coating. Thedelay in release can occur by coating non-active agent containing layerson top of the active agent containing layers. The number of layers canbe adjusted to tune the release of the active agent from the biomaterialcoating.

The active agent can be a cytokine such as IL-4, IL-13, and IL-10 orglucocorticoids such as betamethasone, clocortolone, cortisone,dexamethasone, fludrocortisone, fluocortolone, fluprednylidene,hydrocortisone, medrysone, methylprednisolone, paramethasone,prednisolone, prednisone, prednylidene, triamcinolone, triamcinoloneacetonide and their esters. In certain non-limiting embodiments, asexemplified below, the active agent can be IL-4. In certain embodiments,the cytokine is complexed to the polyelectrolyte, and the concentrationof the cytokine is dependent on the ratio of cytokine complexed with thepolyelectrolyte and the number of active agent containing layers. Forexample, IL-4 can be complexed with dermatan sulfate at a particularratio (e.g., 1.5 μg/mL IL-4 to 2 mg/mL dermatan sulfate). In certainembodiments, the ratio of active agent to dermatan can be from about1:2000 to about 1:10. In certain embodiments, the ratio of active agentto dermatan can be from about 1:1900 to about 1:20, about 1:1800 toabout 1:30, about 1:1700 to about 1:40, about 1:1600 to about 1:50,about 1:1500 to about 1:60, about 1:1400 to about 1:70, about 1:1300 toabout 1:80, about 1:1200 to about 1:90, about 1:1100 to about 1:100,about 1:1000 to about 1:200, about 1:900 to about 1:300, about 1:800 toabout 1:400, or about 1:700 to about 1:500.

The active agent containing layer can include one type of active agentor a combination of different active agents. In certain embodiments,each active agent containing layer contains only one type of activeagent. In certain embodiments, the active agent containing layercontains more than one type of active agent, wherein the differentactive agents are in the same and/or different layers.

In certain non-limiting embodiments, the active agent containing layercontains additional excipients. For example, the active agent containinglayer can contain a polycation and/or polyanion. In certain non-limitingembodiments, as exemplified below, the active agent can be IL-4 incombination with dermatan sulfate.

7.2. Methods of Coating the Biomaterial

The present invention also relates to methods for coating thebiomaterial. In certain non-limiting embodiments, the biomaterial can benegatively or positively charged or treated such that the surfacebecomes negatively or positively charged. The coating process can occurby alternate cyclic deposition of multiple polyelectrolyte layersmediated by opposite electrostatic charges on the surface of a chargedsubstrate.

In certain non-limiting embodiments, the negatively charged biomaterialcan be coated with a polycation layer. In certain embodiments, thepositively charged biomaterial can be coated with a polyanion layer. Incertain non-limiting embodiments, the biomaterial can be coated withalternating polycation and polyanion layers. In certain non-limitingembodiments, once the biomaterial is coated with alternating polycationand polyanion layers, the coated biomaterial can be coated with at leastone layer containing at least one active agent. In certain non-limitingembodiments, polycation and/or polyanion layers can be among and/or ontop of the active agent containing layer(s). In certain embodiments, thecoated biomaterial can be sterilized.

In certain non-limiting embodiments, the surface of the biomaterial iscleaned prior to the addition of a charge or any of the coating layers.For example, the surface of the biomaterial can be cleaned with asolution of water, acetone, isopropanol, ethanol, methanol, benzene,hydrogen peroxide, dioxane, tetrahydrofuran or combinations thereof.

In certain non-limiting embodiments, as exemplified below, thebiomaterial can become charged. The biomaterial can be irradiated toform a consistent and durable charge on the surface of the biomaterial.For example, the biomaterial can be irradiated with radio frequency glowdischarge (RFGD) or plasma-enhanced chemical vapor deposition (PECVD) toform either a negative or positive charge on the surface of thebiomaterial.

In certain non-limiting embodiments, the polycation can be dissolved ina suitable solvent or buffer known to those of skill in the art for theparticular polycation. In certain embodiments, the polyanion can bedissolved in a suitable solvent or buffer known to those of skill in theart for the particular polyanion. Suitable solvent include, but are notlimited to, water and acetate, phosphate, saline buffer, acetic acid,hydrochloric acid, methanol, isopropanol, ethanol, n-propanol,n-butanol, isobutanol, t-butanol, dimethyl sulfoxide, N,N-dimethylformamide, N, N-dimethylacetamide, methyl acetate, ethylacetate, isopropyl acetate, acetone, methyl ethyl ketone, methylisobutyl ketone, or combinations thereof.

The biomaterial can then be soaked in a solution of the polyelectrolyteand then washed in water or buffer and allowed to dry. The coatedbiomaterial can then be soaked in a solution of polyelectrolyte of anopposite charge, washed in water or buffer and allowed to dry. Incertain embodiments, the drying process can utilize pressurized cleanair. This process can continue until the appropriate amount of layers isadded to the biomaterial.

In certain non-limiting embodiments, as exemplified below, thenegatively charged biomaterial can be dipped in a chitosan solution for10 minutes at room temperature, then washed three times in milli-Q waterand air dried, and once dried, the biomaterial can be dipped in adermatan sulfate solution for 10 minutes at room temperature, thenwashed three times in milli-Q water and air-dried.

In certain non-limiting embodiments, once the biomaterial is coated withthe appropriate amount of polyelectrolyte layers, it can be coated withan active agent containing layer. In certain embodiments, the activeagent containing layer is coated on top of the biomaterial without thepolyelectrolyte layers. The coated or uncoated biomaterial can be soakedin the active agent containing solution and then washed in water orbuffer and allowed to dry. Depending on the polyelectrolyte present inactive agent containing layer, the biomaterial is next soaked in apolyelectrolyte solution (with or without an active agent) of theopposite charge and then washed in water or buffer and allowed to dry.This process can continue until the appropriate amount of active agentcontaining layers are added to the biomaterial.

In certain non-limiting embodiments, as exemplified below, thebiomaterial can be dipped in a solution of IL-4-dermatan sulfate mixturefor 10 minutes at room temperature, then washed three times in milli-Qwater and air dried, and once dried, the biomaterial can be dipped in achitosan solution for 10 minutes at room temperature, then washed threetimes in milli-Q water and air-dried.

In certain non-limiting embodiments, the coated biomaterial can besterilized using ethylene oxide gas, gamma irradiation, and E-beamsterilization.

In certain non-limiting embodiments, the method of coating thebiomaterial can be as follows. For this illustration the biomaterial isa polypropylene mesh, but any biomaterial can be used. The method ofcoating the mesh can entail washing the biomaterial with a cleaningsolution, such as, but not limited to water, acetone, isopropanol,ethanol, methanol, benzene, hydrogen peroxide, dioxane, tetrahydrofuranor combinations thereof (e.g., a 1:1 acetone:isopropanol mixture)followed by air drying. The washed mesh can then be further cleaned to,for example, remove any organic contamination by any method known bythose of skill in the art. For example, the washed mesh can beirradiated with gas plasma, such as but not limited to argon plasma(e.g., at 600 W with a gas flow of 35 mL/min with a steady pressure of250 mTorr). Once clean, the mesh can be treated to obtain a negativelycharged surface. For example the cleaned mesh can be exposed to anadapted radio frequency glow discharge (RFGD) via a microwave plasmaprocedure (e.g., maleic anhydride can be used as a monomer for RFGDtreatments followed by hydrolysis). In certain non-limiting embodiments,the mesh can be washed with water and boiled in fresh water prior to thecoating process. In order to deposit a conformal coating onto thesurface of negatively charged mesh, a Layer by Layer (LbL) procedure canbe performed. The charged mesh can undergo alternating immersion intopolycation and polyanion solutions (e.g., 2 mg/mL, 10 minutes each atroom temperature) with intermediate washings in water. For example, thepolycation can be chitosan (dissolved in 0.5% acetic acid for example)and the polyanion can be dermatan sulfate (dissolved in water). First,the mesh can be dipped in the polycation solution for 10 minutes at roomtemperature, then washed (e.g., 3 times—10, 20 and 30 seconds—in milli-Qwater) and air dried (e.g., using pressurized clean air). Next, the meshcan be dipped in a polyanion solution for 10 minutes at roomtemperature. The mesh can then be washed again in milli-Q water andair-dried. This coating cycle can be repeated until a core coating ofbilayers is achieved (e.g., 10). After coating, the mesh can belyophilized and stored at 4° C. Next, the core coated mesh can be coatedwith the active agent. For example, but not by way of limitation, theactive agent IL-4 (e.g., 1.5 μg/mL) can be complexed with dermatansulfate (e.g., 2 mg/mL) by incubating the mixture overnight at 4° C.Then, the coated mesh can be further coated with 20, 40 and 60 bilayerscontaining the active agent using the same LbL method used for the corecoating. After coating, the active agent coated loaded mesh can belyophilized and stored at −20° C.

7.3. Kits

The present invention further provides kits that can be used to practicethe invention. For example, and not by way of limitation, a kit of thepresent invention can comprise a coated biomaterial. In certainembodiments, a kit of the present invention can optionally compriseinstructions on how to use the kit for implanting the coatedbiomaterial.

The present invention further provides kits for preparing the coatedbiomaterial. In certain embodiments, the kit of the present inventioncontains the polycation (in dry or liquid form) and/or polyanion (in dryor liquid form) for application on the biomaterial. When thepolyelectrolyte is provided in dry form, the kit can contain theappropriate buffer or solvent to create the polyelectrolyte solution. Incertain embodiments, the kits can contain the active agent and/orexcipients to create the active agent containing layer. The kits canoptionally contain the biomaterial to be coated and/or instructions forcoating the biomaterial.

The following Example is offered to more fully illustrate thedisclosure, but is not to be construed as limiting the scope thereof.

8. EXAMPLE 1: SHIFTS IN MACROPHAGE PHENOTYPE AT THE BIOMATERIALINTERFACE VIA IL-4 ELUTING COATING 8.1. Introduction

The present example describes the development of a cytokine elutingcoating for transiently shifting macrophage phenotype at thehost-implant interface and demonstrates associated improvements intissue integration of a polypropylene mesh commonly utilized in therepair of pelvic organ prolapse, a procedure associated with high ratesof implant-related complications. In particular, the present exampleexamined whether transient and controlled polarization of macrophages atthe tissue-implant interface towards an anti-inflammatory/regulatory(M2) phenotype during early stages of the host response would mitigatethe chronic foreign body reaction and promote better integration ofpolypropylene mesh into the host tissue in the long-term.

A nanometer-thick coating capable of locally releasing IL-4 (an M2polarizing cytokine) from an implant surface (e.g., mesh in thisexample) in a controlled manner was developed. IL-4 was selected as itis widely used to polarize macrophages to an M2 phenotype in-vitro and aknown driver of macrophage polarization in-vivo (2, 20, 21). Thenanometer thickness of this coating was designed to preserve thearchitecture of the implantable mesh, which is commonly thought to beimportant for adequate tissue in growth and mechanical performance inclinical settings (22, 23). This coating is based on the layer by layer(LbL) technique (24, 25), consisting of an alternate cyclic depositionof multiple polyelectrolyte layers mediated by opposite electrostaticcharges on the surface of a charged substrate (FIG. 1 ). This method haspreviously been shown to produce a tunable, uniform and conformalcoating of nanometer thickness for controlled release of proteins(24-31). Therefore, the number and sequence of layers can be easilymodified in order to provide the desired amount and release time ofIL-4.

Results of XPS, ATR-FTIR and Alcian blue staining confirmed the presenceof a uniform, conformal coating consisting of chitosan and dermatansulfate. Immunolabeling showed uniform loading of IL-4 throughout thesurface of the implant. ELISA assays revealed that the amount andrelease time of IL-4 from coated implants were tunable based upon thenumber of coating bilayers and that release followed a power lawdependence profile. In-vitro macrophage culture assays showed thatimplants coated with IL-4 promoted polarization to an M2 phenotype,demonstrating maintenance of IL-4 bioactivity following processing andsterilization. Finally, in-vivo studies showed that mice with IL-4coated implants had increased percentages of M2 macrophages anddecreased percentages of M1 macrophages at the tissue-implant interfaceduring early stages of the host response. These changes were correlatedwith diminished formation of fibrotic capsule surrounding the implantand improved tissue integration downstream. The results of this exampledemonstrate a versatile cytokine delivery system for shiftingearly-stage macrophage polarization at the tissue-implant interface andsuggest that modulation of the innate immune reaction, rather thanattempts to evade the immune system, may represent a preferred strategyfor promoting biomaterial integration and success.

8.2. Materials and Methods Materials

A polypropylene mesh, Gynemesh® PS (Ethicon, Somerville, N.J.) was used.Maleic anhydride, chondroitin sulfate B, chitosan (low molecular weight,deacetylation degree 85%), chondroitinase ABC, chitosanase, bovine serumalbumin (BSA) and histologic staining materials were supplied by SigmaAldrich (St. Louis, Mo.). Murine IL-4, anti-murine IL-4 antibody, murineIL-4 ELISA detection kit were supplied by Peprotech (Rocky Hill, N.J.).Mouse Arginase-1 antibody (rabbit), anti-rabbit Alexa-fluor 488(donkey), and anti-rabbit Alexa-fluor 594 (donkey) were supplied byAbcam (Cambridge, Mass.). Mouse iNOS antibody (rabbit) was supplied bySanta Cruz (Dallas, Tex.), mouse F4/80 antibody (rat) was supplied byAbD Serotec/Bio Rad (Raleigh, N.C.), and anti-rat Alexa-fluor 488(donkey) and anti-rabbit Alexa-fluor 546 (donkey) secondary antibodieswere supplied by Thermo Fisher (Pittsburgh, Pa.).

Plasma Treatment, Layer by Layer (LbL) Coating and IL-4 Loading ofPolypropylene Meshes

Polypropylene (PP) meshes were cleaned using a 1:1 acetone:isopropanolmixture and then air dried prior to irradiation with 15 seconds of argonplasma at 600 W, an argon gas flow of 35 mL/min and a steady statepressure of 250 mTorr (50 mTorr initial pressure) using an Ion 40 GasPlasma System (PVA Tepla America, Inc).

An adapted radio frequency glow discharge (RFGD) based on a previouslydeveloped microwave plasma procedure was used to obtain a negativelycharged surface (32). Maleic anhydride (MA) was used as a monomer forRFGD treatments followed by hydrolysis. Alternating immersion intochitosan and dermatan sulfate solutions (2 mg/mL, 10 minutes each atroom temperature) with intermediate washings in water was thenperformed. This cycle was repeated until desired number of bilayers wasachieved. IL-4 was incubated with dermatan sulfate prior to the coatingprocedure for IL-4 containing mesh groups.

In particular, maleic anhydride powder (1.5 gr) was placed into a glassplate inside of the machine chamber. 1 cm² pieces of PP mesh were thenplaced around the plate to a distance of 8.5 cm. After an initialpressure of 50 mTorr was reached, 30 seconds of maleic anhydride plasmatreatment was performed at 600 W, an argon gas flow of 35 mL/min and asteady state pressure of 250 mTorr. Finally, in order to remove thephysisorbed maleic anhydride and to hydrolyze the anhydrides and producecarboxylic acid groups (negatively charged at physiological pH), PPmeshes were rinsed for 30 minutes with milli-Q water and then boiled for20 minutes in fresh milli-Q water.

In order to deposit a conformal coating of nanometric thickness onto thesurface of negatively charged PP meshes, a Layer by Layer (LbL)procedure was performed. Chitosan was chosen as polycation and dermatansulfate (chondroitin sulfate B) as polyanion. Chitosan was dissolved in0.5% acetic acid and dermatan sulfate in milli-Q water. Bothpolyelectrolytes were prepared at a concentration of 2 mg/mL. First,meshes were dipped in chitosan for 10 minutes at room temperature, thenmeshes were washed 3 times (10, 20 and 30 seconds) in milli-Q water andair dried (pressurized clean air). Next, meshes were dipped in adermatan sulfate solution for 10 minutes at room temperature. Mesheswere washed again in milli-Q water and air-dried. This cycle wasrepeated until a core coating of 10 bilayers was achieved. Aftercoating, meshes were lyophilized and stored at 4° C.

Prior to IL-4 loading onto the meshes, an IL-4 (1.5 μg/mL)-dermatansulfate (2 mg/mL) mixture was made and incubated overnight at 4° C. inorder to complex IL-4 into the polyanion. Then, polypropylene mesheswith a 10-bilayer core coating were further coated with 20, 40 and 60bilayers containing IL-4 (PP⁻[CH/DS]₁₀[CH/DS^(IL-4)]_(x), where x standsfor the number of bilayers and DS^(IL-4) stands for dermatansulfate-bound IL-4). After coating, IL-4 loaded meshes were lyophilizedand stored at −20° C. Coated (no IL-4) meshes were used as controls,using the same numbers of bilayers used for IL-4 loaded meshes. All meshmaterials were then terminally sterilized using ethylene oxide.

In-Vitro and In-Vivo Studies

Coating characterization. An alcian blue staining was performed to stainthe GAG components and reveal the coating. A 1% alcian blue solution wasmade on 3% acetic acid and adjusted to pH 2.5. Coated meshes andcontrols were re-hydrated in distilled water and then immersed into thealcian blue solution for 30 minutes at RT. Then meshes were washed inrunning tap water for 5 minutes and rinsed 5 minutes in distilled water.Images were taken using a standard optical camera.

Additionally, elemental composition of the coated meshes was performedusing an X-ray photoelectron spectroscopy (XPS), using an ESCALAB 250Xi,Thermo Scientific (Pittsburgh, Pa.). To identify the elements in thecoating/surface of the meshes, an initial survey of 10 scans wasobtained and for detailed elemental information, spectra of 25 scanswere obtained for Carbon, Oxygen, Nitrogen and Sulfur. Spectra data wasanalyzed using Advantage software, Thermo Scientific.

Finally, meshes were analyzed under Fourier transform infraredspectroscopy with attenuated total reflectance (ATR-FTIR) using a BrukerVertex 70 (Billerica, Mass.) equipped with a germanium ATR crystal at aresolution of 1 cm⁻¹, 2 mm of aperture, 32 scans and processed by OPUSsoftware to adjust the baseline, to smooth spectra and to remove H₂O andCO₂ peaks due to environmental noise.

IL-4 loading and release assays. Immunolabeling was used toqualitatively corroborate the loading of IL-4 into the coating. IL-4loaded, coated (no IL-4) and pristine meshes were immersed in a 1% BSAsolution to block non-specific adsorption of antibodies (1 h, RT).Washing was performed in between each step by dipping the meshes 4 timesin 0.05% Tween 20. Then meshes were immersed and incubated in a solutionof anti-murine IL-4 (from rabbit) as primary antibody (1:100 in 0.1%BSA, 2 hours, RT). Later on, meshes were immersed in a solution ofanti-rabbit-Alexa Fluor 546 as a secondary antibody (1:100 in 0.1% BSA,30 min, RT). Mesh fluorescence was observed under confocal microscopy(Leica DMI4000 B, Buffalo Grove, Ill.), in which an excitation/emissionof 480/520 nm has been used to observe the mesh autofluorescence (green)and 561/572 nm to observe the specific fluorescence due to the loadedIL-4 (red).

Loading efficiency and release assays were performed followingmanufacturer instructions of Peprotech IL-4 ELISA kit. First, 1 cm²pieces of IL-4 loaded (20, 40 and 60 bilayers) and coated (no IL-4)meshes were immersed into 400 μL of a solution 0.05 units/mLchondroitinase ABC and 0.05 units/mL chitosanase in 1×PBS. Incubationwas performed to multiple time points at 37° C., after which 400 μL ofsolution were aliquoted and stored at −80° C. until the end of theexperiment. After collection, replacement with fresh solution wasperformed to continue the release assay. To perform the ELISA assays,100 μL aliquots were used from each sample (N=9) at each time point.

To determine release profile kinetics; correlation and curve fittinganalyses were performed using the data from cumulative release versustime, until the first time point where the release reaches a plateau,which corresponds to the total release. To corroborate power lawdependence, besides direct curve fitting tests, a linear trend wascorroborated using a LOG (cumulative release) versus LOG (time) curve.

In-vitro macrophage culture assay. An in-vitro macrophage culture assaywas performed in order to demonstrate preservation of bioactivity ofIL-4 released from the coated meshes. Bone-marrow mononuclear cells wereobtained from murine bone marrow as previously described (51), thenthese cells were seeded in plates and differentiated to macrophages withDMEM, 10% FBS, 10% L929 supernatant, 1% HEPES, 2% MEM NEAA, 0.1%β-2-mercaptoethanol (Sigma Aldrich, St. Louis, Mo.) for 7 days. 5×10⁵cells were plated into 24-well plates with α-MEM, 10% FBS, 0.05 units/mLof both chondroitinase ABC and chitosanase. Macrophages were exposed to1 cm² pieces of IL-4 (40 bilayers), coated (no IL-4) and pristinemeshes. Immunolabeling isotype (rabbit IgG) and soluble IL-4 (20 ng/mL)were used as negative and positive controls, respectively. Cells wereincubated at 37° C. and 5% CO₂ for 72 hours. After incubation, cellswere fixed with 2% PFA and then blocked with 2% horse serum, 1% BSA,0.1% triton X-100, 0.1% tween-20 for 1 hour at RT. Immunolabeling wasperformed using anti arginase-1 as primary antibody (1:200, overnight at4° C.) and Alexa Fluor-488 (1:300, 1 hour at RT) as secondary. A 500 nMDAPI solution was used stain nuclei. Images were taken in an array of3×3 images per each well using a Carl Zeiss Observer.Z1 microscope andthen the intensity of arginase-1 staining was analyzed using CellProfiler Image Analysis Software (Broad Institute, Cambridge, Mass.)using the same number of cells for all tested conditions.

In-vivo mouse mesh implantation. An implantation model using C57BL/6female mice, 8-10 weeks old was used, following proper housing andtreatment procedures approved by the Institutional Animal Care and UseCommittee (IACUC) of the University of Pittsburgh and the NationalInstitutes of Health Guide for the Care and Use of Laboratory Animals. Apower analysis was performed to determine that 7 animals per group wererequired to maintain a statistical power of 80%.

Briefly, a midline incision was made and a subcutaneous pocket wascreated in the abdomen of each mouse in order to implant a 1 cm² pieceof IL-4 loaded (40 bilayers), coated (no IL-4) or pristine mesh. PCLsutures were used to close the incision, then 0.5 mg/kg of Baytril and0.2 mg/kg of Buprenex were administered for 3 days as antibiotic andanalgesic, respectively. Buprenorphine (Buprenex), an opioid analgesic,has been studied and shown not to exert any effects or alterations inthe immunological response. After 7, 14 or 90 days; mice were euthanizedand skin/mesh/muscle complex tissues were harvested and fixed for 72hours in neutral buffered formalin. Finally, fixed tissues were paraffinembedded and cross-sections of 7 μm were used for histologicalprocessing for H&E, Masson's Trichrome and Picro Sirius Red staining.

Histological staining. Paraffin embedded tissue cross-sections were usedfor H&E, Masson's trichrome and Picro Sirius Red staining. H&E andMasson's trichrome stained tissue sections were imaged on a NikonEclipse E600 microscope (Tokyo, Japan) at 10× and 20×, respectively.Picro Sirius Red stained tissue sections were imaged at 20× on a NikonEclipse TE2000-E (Tokyo, Japan), equipped with circularly polarizedlight.

ImageJ (version 1.48, NIH) equipped with a color deconvolution plug-in(version 1.5) was used to quantify the area of capsule surrounding meshfibers at 90 days (3 different single fibers per sample, N=7 each group)in images taken from histological tissue sections stained with Masson'sTrichrome.

A custom-designed algorithm (Mathworks MathLab, version R2015a. Natick,Mass.) was used to evaluate quantitatively the quality of the collagencapsule surrounding mesh fibers at 90 days (3 different single fibersper sample, N=7 each group) in images taken from histological tissuesections stained with Picro Sirius Red.

Immunolabeling of histological sections. Paraffin embedded tissuesections were deparaffinized and hydrated in a series ofxylene/alcohol/water. Incubation was performed with proteinase K (1×)for 10 minutes to retrieve antigens. After 3 washes in water, sampleswere incubated at 37° C. in 50 mM of CuSO₄ in 10 mM NH₄Ac buffer (pH=5),to reduce tissue background fluoresence. Slides were washed twice inTBST (25 mM Tris buffer+0.1% tween 20). Then, a 5% donkey serum+2%BSA+0.1% tween 20+0.1% triton X-100 solution was used as blocking agent(2 hours, RT). To immunolabel M2 macrophages, an arginase-1 (1:100) andF4/80 (1:50) primary antibodies were used (overnight at 4° C.), followedby anti-rabbit Alexa Fluor 594 (1:200) and anti-rat Alexa Fluor 488(1:100) secondary antibodies (40 min at room temperature) in blockingbuffer. To immunolabel M1 macro-phages, iNOS (1:100) and F4/80 (1:50)primary antibodies were used (overnight at 4° C.), followed byanti-rabbit Alexa Fluor 594 (1:100) and anti-rat Alexa Fluor 488 (1:100)secondary antibodies (40 min at room temperature) in blocking buffer.Vectashield with DAPI mounting media (Vector laboratories, Burlingame,Calif.) was used to stain nuclei and mount. Images of centered singlefibers (3 different single fibers per sample, N=8 each group) were takenon a Nikon Eclipse E600 microscope equipped with epi-fluorescence at 40×and cell counts were analyzed using ImageJ (version 1.51a, NIH).

Image analysis algorithms were used to quantify the results obtained byimaging of histological tissue sections. First, a custom-designedalgorithm (Wolfram Mathematica, version 10.0. Champaign, Ill.) was usedto quantify both arginase-1 and iNOS expression at 7 and 14 days bymeans of arginase-1/DAPI and iNOS/DAPI pixel ratio versus the distancefrom the surface of single centered mesh fiber (3 different singlefibers, N=8 each group) images taken from histological tissue sectionsper sample. Next image analysis was performed using ImageJ (version1.51a, NIH) in order to quantify the number of pro-inflammatory (iNOS,M1) and regulatory (arginase-1, M2) macrophages (F4/80) surroundingsingle mesh fibers in each group.

Statistical Analysis

Comparisons of means were performed by either one-way or two-wayanalysis of variance (ANOVA), using p<0.05 as statistical significancecriteria (one-tailed) followed by Tukey's test. Shapiro-Wilk was used totest normality. All statistical tests were performed on GraphPad PrismV6 (La Jolla Calif., USA).

8.3. Results Surgical Mesh Plasma Irradiation, LbL Coating andCharacterization

An adapted radio frequency glow discharge (RFGD) method (32) was used toform a consistent and durable negative charge on the surface ofpolypropylene (PP) mesh in order to facilitate the desired LbL coating.The presence of a negatively charged surface was confirmed by theappearance of two peaks at 284 eV (C—C) and 288 eV (O—C═O) on the carbonspectrum and a peak at 532 eV on the oxygen spectrum, while pristinemesh only had a peak at 284 eV (C—C) when evaluated by X-rayphotoelectron spectroscopy (XPS) (FIG. 2A). RFGD treated meshes werethen LbL coated using chitosan as a polycation and dermatan sulfate as apolyanion. Chitosan was chosen for its known biocompatibility,antimicrobial activity, and as activated macrophages highly expresschitinase-like proteins (chitin and chitosan degrading enzymes) (33-35).Dermatan sulfate (also known as chondroitin sulfate B) was chosen forits key role in extracellular matrix (ECM) regulation and its describedability to enhance IL-4 bioactivity in-vivo (36). As such, thechitosan-dermatan sulfate LbL complex was chosen to provide enhancedrelease and bioactivity of IL-4 in the context of macrophage mediatedhost-implant interactions.

A coating of 10 bilayers was performed as core coating prior to IL-4loading. Alcian blue staining was used to visualize the chitosan anddermatan sulfate components of the coating. Blue coloration and absenceof precipitates along the mesh surface suggested the presence of aconformal and uniform coating on LbL coated meshes (FIG. 2B). Electronmicroscopy (FIG. 3 ) was used to confirm the conformal nature of thecoating and showed no apparent changes in surface topography, porosityand thickness between LbL coated, RFGD treated and pristine meshes. Thepresence of chitosan in the LbL coating was corroborated by theappearance of two peaks at 399 eV (C—N) and 401 eV (O—C—N) in thenitrogen spectrum and the presence of dermatan sulfate by the appearanceof a peak at 168 eV (C—S—O) in the sulfur spectrum when evaluated by XPS(FIG. 2A), in addition to the presence of peaks at 288 eV (O—C═O) and286 eV (C—O) in the carbon spectrum, confirming the presence of bothpolyelectrolyte chains. These measurements were performed at differentpoints on the surface of the PP mesh and spectra were identicalthroughout the mesh surface. These findings were consistent withATR-FTIR measurements (FIG. 4 ).

IL-4 Loading, Release and Bioactivity Assessments

Mesh coated with a 10-bilayer core coating was then coated with 20, 40and 60 additional bilayers containing IL-4. IL-4 was pre-incubated withdermatan sulfate prior to LbL coating, promoting the loading of thecytokine due to the high affinity of IL-4 (net positive charge, givenits isoelectric point of 9.17) for sulfated glycosaminoglycans(negatively charged). Confocal microscopy demonstrated positive IL-4labeling distributed throughout the entire surface of IL-4 loaded meshesin contrast to the absence of positive labeling on coated (no-IL-4) meshand pristine mesh (FIG. 5A). ELISA assays were performed to quantifyIL-4 release over time. Results showed that both the amount of IL-4 andthe length of release are dependent on the number of bilayers containingIL-4 in the LbL coating (FIG. 5B). In particular, the in-vitro releaseof IL-4 was observed up to 14, 22 and 30 days for coatings of 20, 40 and60 bilayers, respectively. The release profile for all IL-4 loadedmeshes followed a power law dependence, regardless of the number ofcoating bilayers (FIG. 6 ). These findings are consistent with otherstudies done on LbL films as a platform to study protein release. Basedupon these results, meshes coated with 40 bilayers containing IL-4 wereselected for further in-vitro and in-vivo assays, given the desire torelease IL-4 and polarize macrophages towards an M2 phenotype only atearly stages of the host response, since the coating released about 90%of IL-4 only at early stages of the host response (up to 14 days). Allfurther assays included coated (40 additional bilayers with no IL-4) andpristine mesh groups as control groups.

In order to show that IL-4 bioactivity remained after the coatingprocedure and terminal sterilization (by ethylene oxide), an in-vitromacrophage polarization assay was performed using bone marrow-derivedmacrophages. Macrophages exposed to IL-4 loaded meshes for 72 hours werefixed and immunolabelled against arginase-1, an M2 macrophage specificmarker. Image analysis (CellProfiler, Broad Institute, Cambridge, Mass.)of arginase-1 positive cells (FIG. 7A) revealed that the IL-4 releasedfrom the IL-4 loaded mesh remained bioactive and able to polarizemacrophages towards an M2 phenotype (FIG. 8 ). No significant increaseof arginase-1 was observed for coated mesh compared to pristine mesh. Ofnote, the pattern of arginase-1 expression following exposure to IL-4coated meshes was similar to the IL-4 positive control (20 ng/mL)despite the lower levels of IL-4 (2.25 ng/mL) released from the meshsurface at 72 h (FIG. 7B), suggesting that the coating components mayenhance IL-4 bioactivity or that IL-4 is protected by the coating andreleased gradually.

Studies on Macrophage Polarization and the Early-Stage Host ResponseAgainst Implanted Mesh

A mouse implantation model was used to test the ability of IL-4 loadedmesh to promote an early shift (7 and 14 days) in the polarization ofmacrophages towards an M2 phenotype in-vivo and to examine the effectsof such shifts in macrophage polarization upon downstream tissueremodeling (90 days). 1 cm² of IL-4 loaded mesh (40B), coated mesh (noIL-4), or pristine mesh were implanted into a subcutaneous pocket in theabdomen of 8-10 week old female C57BL/6J mice. Mesh and surroundingtissue (muscle and skin) were then harvested at 7 and 14 dayspost-implantation and used to study macrophage polarization. Shamsurgeries (no mesh implantation) were also performed. In sham animals, anormal wound healing process observed (FIG. 9 , top panel) and wascharacterized by a transient inflammatory response including significantimmune cell infiltration at 7 days which was largely resolved by 14 dayspost-inflammation with restoration of normal tissue architectureresembling healthy tissue controls. The histologic appearance in miceimplanted with mesh was also characterized by the presence ofinflammatory cell infiltration in the surgical site at 7 days; however,this reaction was not resolved at 14 days and was largely localized tothe area surrounding mesh fibers, regardless of mesh type (FIG. 9 ,bottom panel), thereafter. The presence of foreign body giant cells wasnoted beginning at 14 days post implantation and at the 90 day timepoint, regardless of mesh type. While the number and distribution offoreign body giant cells was qualitatively similar across all groups, noattempt was made in the present study to quantify the number of foreignbody giant cells.

Immunolabeling of F4/80 (pan macrophage marker), arginase-1 (an M2marker) and inducible nitric oxide synthase (iNOS, an M1 marker) wasperformed to assess the number, location, and phenotypic profiles of themacrophages within the site of implantation at 7 and 14 dayspost-implantation. Image analysis was performed using a custom-designedalgorithm (Wolfram Mathematica, Version 10.0) in order to quantifylabeling (normalized and expressed as cumulative arginase-1/DAPI pixelratio) as a function of distance from the mesh surface (FIG. 10 ). Inall mesh groups, the number of both arginase-1 and iNOS positive cellswere observed to peak within the first 50 μm from the mesh surface(FIGS. 10B and 10D). Therefore, this distance was considered as thetissue-biomaterial interface, where the most important interactions ofthe biomaterial with the surrounding tissue occur and determine theimplant success in the long term.

Total cell infiltration around single mesh fibers was assessed by DAPIstaining, revealing no differences between groups at 7 or 14 days (FIG.11A). However, the small increases in the number of cells within theremodeling site were observed from 7 to 14 days in the pristine and IL-4loaded mesh implantation groups. Analysis of F4/80 positive macrophagepopulations revealed a significantly higher presence of F4/80 positivecells as a percentage of the total cell population in mice implantedwith pristine mesh, compared to both coated (no IL-4) and IL-4 loadedmesh groups at 7 days (FIG. 11B). At 14 days, the percentage of F4/80positive cells in the pristine mesh group was significantly reduced andwas similar to levels similar to those found in both coated (no IL-4)and IL-4 loaded meshes. The percentage of F4/80 positive cells in theimplantation site of IL-4 loaded meshes were also significantlydecreased compared to 7 days, but these decreases were smaller thanthose observed for the pristine mesh group. There were no differences inthe percentage of F4/80 positive cells between coated (no IL-4) and IL-4loaded mesh groups at 7 or 14 days. These results suggest that thecoating may have had an inhibitory effect upon the recruitment ofmacrophages into the implantation site at early time points.

Additional co-labeling was performed for arginase-1 and iNOS to assessthe M1/M2 polarization profile of the cells within the implantationsite. Results at 7 days post-implantation revealed that mice implantedwith IL-4 loaded mesh had an increase in the percentage of arginase-1positive macrophages (F4/80⁺) near the mesh surface as compared tocoated mesh and pristine mesh groups (FIG. 11C). The number ofarginase-1 positive cells in the IL-4 loaded mesh group wassignificantly increased in the first 40 to 50 μm from the mesh surface(FIGS. 10B and 10C) as compared to both coated and pristine mesh,suggesting that the effects of IL-4 released from the LbL coating arelimited to distances up to 50 μm from the surface of the implanted mesh.Coated mesh did not elicit a significant increase in arginase-1 positivemacrophages as compared to pristine mesh (FIG. 11C). These results areconsistent with the in-vitro findings showing significant increases inM2 macrophage polarization only in the IL-4 loaded mesh group.Similarly, results at 7 days post-implantation also showed a reductionof iNOS positive cells in mice implanted with IL-4 loaded meshescompared to mice implanted with pristine meshes (FIG. 11D). Miceimplanted with coated mesh also showed a reduction in iNOS positivemacrophages compared to the pristine mesh implanted group; however, nosignificant differences were observed between the coated mesh and IL-4loaded groups (FIG. 8D). These results suggest that the coating materialmay have impacted the polarization of macro-phages towards an M1profile. Differences in iNOS labeling were observed to peak at 25 μmfrom the mesh surface of the pristine mesh implanted group at 7 days(FIG. 10E), again suggesting that the effects of the coating werelimited to the first 50 μm from the mesh surface.

Results at 14 days post-implantation revealed a decrease in botharginase-1 and iNOS labeling as compared to 7 days, with no significantdifferences observed between any groups. However, the percentage ofarginase-1 positive macro-phages was still higher than both coated (noIL-4) and pristine mesh groups (FIG. 11C). The percentage of iNOSApositive macrophages at 14 days was found to decline in mice implantedwith pristine mesh as compared to 7 days; however, there were nosignificant differences observed between any groups at the 14 day timepoint (FIG. 11D). When the effects of IL-4 coating upon arginase-1expression at 7 and 14 days were compared (FIGS. 10C, 10F, 11D) it canbe appreciated that arginase-1 expression in the IL-4 coated group at 14days returned to levels similar to those observed for pristine meshes atboth 7 and 14 days. This suggests that the length of IL-4 release fromthe LbL coated meshes occurs at the early stages of the host response(<14 days), and that its effects on macrophage polarization in-vivo aredeclining by 14 days. Expression of iNOS in the IL-4 loaded mesh groupremained low with no changes between 7 and 14 days.

While increases in the M2 macrophage population can likely be attributedto the release of IL-4 from loaded mesh as demonstrated in-vitro, thereare two possible mechanisms that could explain the observed reduction inthe number of iNOS positive cells in the IL-4 loaded mesh group. First,iNOS expression may be reduced as a consequence of the polarization ofthe macrophages at the tissue implant interface towards an M2 phenotype,given the known competitive nature of pathways leading to iNOS andarginase-1 expression in mice (37-39). Second, decreased iNOS expressionmay be due to effects of the coating components upon macrophagepolarization, and hence a diminished M1 macrophage response. This secondmechanism is supported by the significant reduction in iNOS positivemacrophages (FIG. 11D) and also the reduction in F4/80⁺ cells (FIG. 11B)observed in the coated mesh group at 7 days, compared to the pristinemesh group (FIG. 10F). Therefore, the coating components and/or themodified mesh surfaces themselves appear to have effects in thereduction of M1 macrophages but not in promoting M2 macrophagepolarization. Thus, the observed results are likely a combination ofmechanisms driving the reduction of M1 macrophages by IL-4 loaded mesheswith IL-4 mediated increases in the M2 population.

It was noted that some arginine-1 positive and iNOS positive cells didnot express F4/80. This suggests that cells other than macrophages mayproduce arginine-1 and iNOS in the area of implantation, or that apopulation of macrophages that express other markers such as CD11 b orCD68, but not F4/80, are present within the remodeling site.

Downstream Effects in the Host Response Upon Macrophage PolarizationPromoted by Implanted Meshes

Finally, mesh and the surrounding tissue complex were harvested at 90days post-implantation to evaluate the effects of mesh coating and IL-4loading upon long-term tissue remodeling outcomes. Image analysis ofMasson's trichrome stained histological sections was performed toidentify and quantify capsule formation. Results revealed capsuleformation around mesh fibers for all groups (FIG. 12A); however, IL-4loaded mesh elicited reduced capsule density compared to the prominentand dense capsules surrounding fibers of both coated and pristine meshes(FIGS. 12B and 13 ). Subsequent analysis of collagen fiber distributionin picrosirius red stained sections was performed using acustom-designed algorithm (Mathworks MatLab R2015a) to assess thequality of the collagen fibers composing the fibrotic capsule.Circularly polarized light microscopy was able to reveal the relativethickness of the collagen fibers as a function of the color hue fromthin green fibers to increasingly thick yellow, orange and red fibers(40). Results revealed that mice implanted with IL-4 loaded meshes hadreduced content of both thick orange and thicker red collagen fibers,compared to both pristine and coated meshes (FIGS. 12C and 12D). Aconcurrent increase in thin yellow and thinner green collagen fibers wasfound for IL-4 loaded mesh compared to both pristine and coated mesh(FIGS. 12C and 12D). These outcomes indicate a change in the quantityand type of the collagen fibers composing the fibrotic capsule and maybe particularly relevant for an improved mechanical performance of theimplanted mesh in-vivo.

8.4. Discussion

The results of this example demonstrate that the effects of the releasedIL-4 from LbL coated mesh caused a shift in early-stage macrophagepolarization that was associated with positive long-term effects such asminimized capsule formation and/or improved tissue quality andcomposition as compared to coated and pristine meshes. These resultsalso suggest that long-term positive outcomes are due to an increase inthe proportion of M2 macrophages, rather than a decrease in the presenceof M1 macrophages, given that coated meshes were capable ofsignificantly decreasing the proportion of M1 macrophages (FIG. 10 ) ascompared with pristine mesh, but were not associated with improvedtissue remodeling outcomes (FIGS. 12A-12D). The present studydemonstrated that it is possible to transiently shift the early phasesof the host response to implants which otherwise elicit a chronicpro-inflammatory response with impact upon the tissue remodeling outcomedownstream while leaving key implant characteristics such as materialproperties and porosity intact.

It has been previously suggested that excessive long-term polarizationtowards either an M1 or an M2 phenotypes may have negative effects onremodeling outcomes (3, 13, 41). Additionally, studies have describedpathologies associated with an imbalance and long-term presence of M1 orM2 macrophages, including but not limited to cancer, diabetes andatherosclerosis (12, 42, 43). Therefore, localized and temporal deliveryof bioactive agents represents an advantage over strategies promotingsystemic and or permanent shifts in the host response as it limits thepotential for adverse long-term interactions and exacerbation ofconditions which may exist at distant sites. Similarly, promotingtransient shifts in macrophage polarization in the early host responserepresents an improved approach as compared to strategies which seek toevade the host immune response. Previous studies using surfacemodification of biomaterials and coatings to escape the innate immunesystem have shown only modest improvements at early stages of the hostresponse against biomaterials and few improvements in long-termperformance (44-48). Finally, the present delivery system represents anadvantage over previous delivery approaches, given that significanteffects on macrophage polarization are observed at lower, controllableand safer doses (picograms to nanograms), compared to the high doses(nanograms to micrograms) of IL-4 used in previous studies. The systemicrelease of larger amounts IL-4 may lead to effects upon distal tissuesand/or exacerbated and contradictory outcomes associated with a fibroticprocess or potential enhancement of the foreign body reaction.

In-vivo, both the chitosan and dermatan sulfate components of thecoating are degraded by macrophages and other cells participating in thehost response. Layer by layer films have shown multiple releasemechanisms, depending the nature of the polyelectrolytes composing thefilms, surface degradation being the most predominant mechanism thatgradually releases the entrapped bioactive agents by degradation of themost external layer films, followed by more internal layers. Therefore,IL-4 can be released by gradual surface degradation of the coatingmultilayers, mainly triggered by macrophages and other cells of theimmune system with enzymatic capacity. However, release by diffusion ofIL-4 can also occur to a lesser extent.

In sum, the presence of a uniform and conformal coating composed of bothchitosan and dermatan sulfate was demonstrated. This coating can beloaded with IL-4 in a uniform manner through the entire surface of themesh, and the amount and length of release can be tuned by simplychanging the number and sequence of coating bilayers. The released IL-4from LbL coated meshes is bioactive and can promote macrophagepolarization towards an M2 phenotype both in-vitro and in-vivo. Inaddition, the effects of the local released IL-4 from LbL coated mesheson macrophage polarization extend up to 50 μm of distance from the meshsurface at early stages of the host response against biomaterials.Consequently, these effects also led IL-4 loaded meshes to reduce thepercentage of M1 macrophages in-vivo, compared to pristine meshes. Atlong term, a decreased fibrotic capsule formation surrounding meshfibers and an improved quality of collagen fibers composing the capsuleobserved only in mice implanted with IL-4 loaded meshes indicates animproved resolution of the foreign body reaction. These positivelong-term outcomes are mostly attributed to an increased proportion ofM2 macrophages rather than a reduced presence of M1 macrophages.

Finally, these results support our hypothesis that early-stagemacrophage polarization at the tissue-implant interface towards an M2phenotype would mitigate the foreign body reaction and hence promotebetter integration of the mesh into the host tissue in the long term,compared to the outcomes observed from the passive and non-controlledeffects on macrophage polarization promoted by different biomaterials.This example also demonstrated that strategies which include modulationof the macrophage response are beneficial for tissue integration andfunctional remodeling of implanted biomaterials, rather than strategieswhich simply seek to avoid the innate immune system. While the presentstudy focused only upon polypropylene mesh commonly used for soft tissuereconstruction, the methods and findings presented can be extended toinclude other material types and applications.

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The invention claimed is:
 1. A kit for remodeling a target tissue,comprising: a biomaterial coated with a coating, wherein the coatingincludes (a) at least one polycation layer; (b) at least one polyanionlayer; (c) at least one active agent-containing layer comprisingdermatan sulfate and an M2 polarizing active agent, wherein the M2polarizing active agent is pre-incubated with the dermatan sulfate in aratio between about 1:10 to about 1:2000 to be complexed, and whereinthe at least one polycation layer and the at least one polyanion layeralternate to form a bilayer; and instructions for implanting thebiomaterial adjacent to the target tissue.
 2. The kit of claim 1,wherein the instructions comprise information for implanting thebiomaterials to release an effective concentration of the M2 polarizingactive agent at a target tissue-biomaterial interface.
 3. The kit ofclaim 1, further comprising additional polycation and polyanion fortuning a number or a composition of the polycation layer, the polyanionlayer, the active agent-containing layer, or a combination thereof. 4.The kit of claim 1, wherein the biomaterial is selected from the groupconsisting of mesh, polypropylene, metal, tissue engineering scaffold,polytetrafluoroethylene, polyethylene, polystyrene, and combinationsthereof.
 5. The kit of claim 1, wherein polycation in the at least onepolycation layer is selected from the group consisting of apolysaccharide, a protein, a synthetic polypeptide, a syntheticpolyamine, a synthetic polymer, and combinations thereof.
 6. The kit ofclaim 1, wherein polyanion in the at least one polyanion layer isselected from the groups consisting of a polysaccharide, a protein, asynthetic polypeptide, a synthetic polyamine, a synthetic polymer, andcombinations thereof.
 7. The kit of claim 1, wherein polycation in theat least one polycation layer is chitosan, and the polyanion in the atleast one polyanion layer is the dermatan sulfate.
 8. The kit of claim1, wherein the M2 polarizing active agent is IL-4.
 9. The kit of claim1, wherein the coating is from about 0.5 nm to about 500 μm.
 10. The kitof claim 1, wherein the biomaterial comprises a total of about 4 toabout 20 bilayers, wherein the bilayers do not contain the M2 polarizingactive agent.
 11. The kit of claim 1, wherein the coated biomaterialcomprises about 20 to about 100 M2 polarizing active agent containinglayers.
 12. A kit for coating a biomaterial, comprising: a polycationfor coating the biomaterial with a polycation layer; a polyanion forcoating the biomaterial with a polyanion layer; an M2 polarizing activeagent and dermatan sulfate to be incorporated into at least one layer,wherein the M2 polarizing active agent is pre-incubated with thedermatan sulfate in a ratio between about 1:10 to about 1:2000 to becomplexed; and instructions for preparing a coated biomaterial, whereinthe coated biomaterial comprises the polycation layer and the polyanionlayer that alternate to form at least one bilayer.
 13. The kit of claim12, further comprising the biomaterial to be coated.
 14. The kit ofclaim 13, wherein the biomaterial is selected from the group consistingof mesh, polypropylene, metal, tissue engineering scaffold,polytetrafluoroethylene, polyethylene, polystyrene, and combinationsthereof.
 15. The kit of claim 12, wherein the polycation and thepolyanion are in dry form.
 16. The kit of claim 15, further comprising abuffer for creating a polycation solution and a polyanion solution. 17.The kit of claim 12, wherein the polycation and the polyanion are inliquid form.
 18. The kit of claim 12, wherein polycation is selectedfrom the group consisting of a polysaccharide, a protein, a syntheticpolypeptide, a synthetic polyamine, a synthetic polymer, andcombinations thereof.
 19. The kit of claim 12, wherein polyanion isselected from the groups consisting of a polysaccharide, a protein, asynthetic polypeptide, a synthetic polyamine, a synthetic polymer, andcombinations thereof.
 20. The kit of claim 12, wherein the instructionsinclude information for layer by layer coating of the biomaterial usingthe polycation, the polyanion, the M2 polarizing active agent, and thedermatan sulfate.